Summary/Abstract: People may be exposed to pesticides through various occupational and environmental routes, resulting in numerous negative health effects. Measuring chemical exposures in human beings provides the most accurate estimation of a person?s true exposure level. However, a major impediment towards precisely assessing exposure level has been the lack of affordable technologies that can readily and sensitively measure exposure biomarkers using minimally invasive fluids (i.e. saliva, drop of blood or urine). In this SBIR project, DL ADV-Tech proposes to develop an ultra-sensitive, portable, inexpensive and accurate sensor platform capable of non- invasively quantifying individual?s pesticide exposures using urine as relevant biological fluids. The proposed biosensor includes a multi-channel test-strip, where advanced nanomaterial (single-atom nanozyme) is introduced to achieve high sensitivity, and a portable smartphone-based reader to provide accurate analysis and instant data collection. Single-atom nanozyme is a new type of nanomaterial that exhibits enzyme-like properties, which possessed remarkable catalytic activity comparable to natural enzymes, high robustness under harsh conditions, superior ?pH and ?thermal stability, low cost and easy mass preparation. This new type of enzyme- like nanomaterial makes this proposed sensor strategy substantially more compelling for use in detecting low level exposure concentrations. Of particular importance to this proposal is the ability to couple a simple sample separation strategy with a parallel multiplex sensor platform for simultaneous target analysis and to increase sensitivity using single-atom nanozyme enhanced biosensors over more conventional enzyme-linked immunosorbent assay (ELISA). Meanwhile, the smartphone-based reader, which will be developed based on our compact design and advanced analysis algorithm, will provide accurate analysis and enable measurement across multiple stressors and scales. Using the proposed biosensor, the measurement of target exposure can be completed within 10 minutes and the detection limit can as low as 0.1 ng/ml. The cost for each measurement will be less than $1. The proposed biosensor will allow sensitive, accurate, non-invasive, and low cost analysis of pesticide exposures, which will provide a revolutionary technique to monitor exposure level in population. The proposed biosensor will enable acquisition of comprehensive information for understanding pesticide risks and mitigating their threat to public health. There is a large market and significant commercial opportunity for such effective technique. A key focus of current proposal in Phase 1 is to develop and refine the proposed biosensor for quantification of exposure levels of 2,4-D and glyphosate, and demonstrate sensitivity, accuracy and on-site analysis in human urine samples. This platform will enable a more accurate prediction of exposures and once validated, can be employed to assess pesticide mixtures, which is of a great importance in public health and personal exposure in population studies. 1